Topoisomerases are ubiquitous proteins found across all three domains of life (bacteria, archaea, and eukarya). They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. Topoisomerases change the topology of DNA by transiently breaking one (type I) or two (type II) DNA strands and passing another single or double strand through the break. The study of the structure and function of topoisomerases promises not only to further our understanding of proteins that interact with DNA and alter its topological properties, but also to provide important information to aid in the design of new therapeutic agents. This proposal is concerned with biophysical and structural studies of different topoisomerases. The long term goal of the project is to provide a comprehensive understanding of topoisomerase action at many different length scales, from the atomic level to the nano scale. In the past period we made substantial progress towards this goal, including solving atomic structures of topoisomerase III in complex with DNA, a structure of D. radiodurans topoisomerase IB in complex with DNA that revealed the existence of a secondary site, structures of several fragments of topoisomerase V, and single molecule studies of topoisomerase V. In addition, we continue making progress with our single molecule studies of topoisomerases and also initiated work on a complex of gyrase with a large DNA fragment. Our studies are providing important information on these molecules and allowing us to relate the atomic structures to the wealth of existing functional, biochemical, and biophysical data. For the next project period we propose to continue and expand our studies of topoisomerases. The specific aims for this proposal are: i) to study the mechanism of type IA topoisomerases at the single molecule level, ii) to study the structure and catalytic mechanism of topoisomerase V, the topoisomerase most recently discovered, and iii) to study the structure of a complex of gyrase with DNA. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the macromolecules that we require for our work, X-ray crystallography and electron microscopy methods to solve their structures, and single molecule studies to elucidate their mechanism.